The present invention relates to compositions and methods for releasing nucleic acid molecules from solid matrices. The invention further relates to compositions and methods for purifying and isolating nucleic acid molecules from biological materials such as animal tissues and plant matter.
It is desirable in many instances to generate and analyze nucleic acid molecules samples obtained from numerous individual entities and/or organisms of populations. Often, such samples are used to identify the genotypes of individuals which reside either in the same or different geographic locations. Thus, the collection and analysis of samples are often employed to determine the genotypes of individual members of populations. Such analyses generally result in the generation of data relating to both the individuals from which the samples are obtained and the populations as a whole.
The collection and analysis of samples which contain nucleic acid molecules from populations of organisms are often performed to obtain genotype data from viral, plant and animal populations. One example of a situation where genotype analysis of large numbers of individuals of members vof populations is commonly performed is where data regarding the genotypes of plants in a geographic location is sought. These data may be generated to determine the spread rate of particular plant strains or to identify genetically modified plants which either have been grown from seeds sold to farmers or are the progeny of plants grown from such seeds.
A number of companies currently sell genetically modified plants and seeds derived from these plants. In some cases, these seeds are sold under the condition that the purchasers, generally farmers, repurchase seeds from their suppliers instead of growing plants from seeds which are obtained from plants grown from purchased seeds. Further, a number of consumer groups, as well as governmental organizations, have objected to the sale of agricultural products prepared from genetically modified plants.
In each instance described immediately above, genotype analyses can be performed to identify genetically modified plants. Such analyses often begin with the collection of large numbers of plant samples obtained in rural settings. Thus, there is a need for methods which allow for the collection and convenient storage of large numbers of samples containing nucleic acid molecules derived from plants which can then be used for genotype analyses.
In other situations, genotype analyses are performed on samples derived from animals (e.g., humans) to generate data related, again, to either individuals or populations of which these individuals are members. Further, genotype analyses performed on samples derived from either animals or plants may be used to obtain data relating to entities associated with these organisms. Examples of such associated entities include viruses such as Human Immunodeficiency Viruses (HIVs). In particular, genotype analyses of HIV populations can be performed using nucleic acid molecules obtained from human blood samples. Due to the rapid rate with which HIVs alter their genomes, genotype analyses have been employed to track the spread and regional predominance of various viral strains.
The use of filter paper (e.g., Whatman 3MM filter paper) provides an inexpensive method for the collection, shipment, and storage of samples which contain nucleic acid molecules (e.g., RNA, plasmids, viral vectors and chromosomal DNA). This is especially the case when samples are collected in remote areas where there is no access to refrigeration.
One example, of a filter paper based medium used for the collection, shipment, and storage of blood samples is FTA(copyright) paper, which is composed of cellulose material impregnated with (i) a monovalent weak base; (ii) a chelating agent; (iii) an anionic detergent; and, optionally, (iv) uric acid or a urate salt. FTA(copyright) paper can be used to store human genomic DNA, for example, in the form of dried spots of whole blood, the cells of which lyse after making contact with the paper. Stored at room temperature, genomic DNA on FTA(copyright) paper is reported to be stable for at least 7.5 years. (Burgoyne et al., CONVENTIONAL DNA COLLECTION AND PROCESSING: DISPOSABLE TOOTHBRUSHES AND FTA(copyright) PAPER AS A NON-THREATING BUCCAL-CELL COLLECTION KIT COMPATIBLE WITH AUTOMATABLE DNA PROCESSING, 8th International Symposium on Human Identification, Sep. 17-20, 1997.) Thus, the placement of nucleic acid samples on filter paper (e.g., FTA(copyright) paper) offers a compact archival system compared to glass vials or plastic tubes located in precious freezer space.
DNA from blood spots has been used in newborn screening programs to identify genetic mutations implicated in several diseases and to provide a means for identifying military personnel. (See, e.g., Seltzer et al., Biochem. Med. Metab. Biol. 46:105-109 (1991); Jinks et al., Hum. Genet. 81:363-366 (1989); Skogerboe et al., Clin. Chem. 37:454-458 (1991); McEwen et al., Am. J. Hum. Genet. 55:196-200 (1994).)
The storage of blood samples on dried filter paper has the additional advantage of pathogen inactivation. More specifically, HIV, as well as a number of other infectious agents, are believed to lose viability upon drying. Further, nucleic acid molecules obtained from these dried blood spots, as well as other dried samples containing nucleic acid molecules, can also be used to isolate and reverse transcribe messenger-RNA (mRNA).
The spotting of bacterial nucleic acids on filter paper can also be used as part of a sample storage and retrieval system. Recently, Rogers and Burgoyne characterized samples of several bacterial strains of Staphylococcus and Escherichia coli stored on FTA(copyright) paper by PCR-ribotyping. (Rogers et al., Anal. Biochem. 247:223 (1997).)
Before analysis of nucleic acids captured by filter papers, washing steps generally need to be performed to remove stabilizing chemicals, if present, and cellular inhibitors of enzymatic reactions. Since DNA, for the most part, remains with the paper through these washing steps, manipulations to purify such nucleic acids are simplified and amenable to automation.
Several methods have been developed for releasing nucleic acids from materials such as FTA(copyright) paper. For example, Burgoyne demonstrated that purified plasmid DNA, stored on paper encased in polystyrene, can be recovered using a uric acid solution. (Burgoyne, U.S. Pat. No. 5,496,562, the entire disclosure of which is incorporated herein by reference.) Another method for nucleic acid release employs a buffer containing a chelating agent in an aqueous solution. (See, e.g., PCT Publications WO 99/39010, WO 99/38962, and WO 99/39009, each of which is incorporated herein by reference.)
The invention provides methods for releasing DNA from solid matrices which are relatively simple in comparison to methods currently in use in the art. Further, the DNA released by methods of the invention can be used directly in a number of processes (e.g., genotyping analyses).
The present invention relates to compositions and methods for the removal of nucleic acid molecules (e.g., DNA) from solid matrices. In particular, the methods of the invention employ releasing reagents to facilitate the release of nucleic acid molecules. The invention further provides compositions relating to these methods.
The present invention also relates to methods for purifying and/or isolating nucleic acid molecules.
In one general aspect, the invention provides methods for removing nucleic acid molecules from solid matrices comprising contacting the solid matrices with releasing reagents which comprise one or more alkanol amines.
In another general aspect, the invention provides methods of purifying and/or isolating nucleic acid molecules comprising:
(a) contacting the nucleic acid molecules with solid matrices under conditions which favor adherence, attachment, association, and/or binding (covalently or non-covalently) of the nucleic acid molecules to the solid matrices: and
(b) contacting the solid matrices containing the bound nucleic acid molecules with releasing reagents comprising one or more alkanol amines, under conditions which favor release of the nucleic acid molecules from the solid matrices. In a related aspect, the methods of the invention further comprise collecting the releasing reagents containing nucleic acid molecules which have been released from the solid matrices.
In specific embodiments, solid matrices used in methods of the invention comprise compounds that prevent or inhibit degradation of nucleic acid molecules, such as one or more weak bases, one or more chelating agents, one or more anionic surfactants, one or more anionic detergents, uric acid, and/or one or more urate salts.
In other specific embodiments, solid matrices used in the methods of the invention are cellulose based matrices or micromesh synthetic plastic matrices. In specific embodiments of the invention, the solid matrix is either a filter paper (e.g., Whatman 3MM paper) or an FTA(copyright) paper.
In yet other specific embodiments, the alkanol amine present in releasing reagents used in methods of the invention comprises an ethanolamine. In particular, the ethanolamine may be mono-ethanolamine, di-ethanolamine, or tri-ethanolamine.
In related embodiments, releasing reagents used in methods of the invention comprise more than one ethanolamine (e.g., two or three ethanolamines).
In certain embodiments, releasing reagents used in methods of the invention are aqueous solutions.
In specific embodiments, releasing reagents used in the methods of the invention have a pH which falls within the range of from about 8.3 to about 13 or from about 10 to about 12. In specific embodiments of the invention, the releasing reagents have a pH of about 11.
In other specific embodiments of the invention, the one or more alkanol amines are present in the releasing reagents at concentrations of from about 0.01% to about 5% (vol./vol.), from about 0.01% to about 3% (vol./vol.), from about 0.01% to about 1% (vol./vol.), or from about 0.1% to about 1% (vol./vol.).
In additional embodiments, solid matrices which contain nucleic acid molecules are incubated with releasing reagents for a time period ranging from about 1 to about 180 minutes, from about 1 to about 120 minutes, from about 10 to about 60 minutes, or from about 10 to about 30 minutes.
In further embodiments, solid matrices which contain nucleic acid molecules are incubated with releasing reagents at about 65xc2x0 C. to about 100xc2x0 C. or about 90xc2x0 C. to about 100xc2x0 C.
In certain embodiments, the methods of the invention comprise separating released nucleic acid molecules from the releasing reagents.
In additional embodiments, nucleic acid molecules released from solid matrices using methods of the invention comprise vectors (e.g., plasmids, artificial chromosomes, etc.). Similarly, nucleic acid molecules released from solid matrices using methods of the invention may comprise nucleic acid molecules of cells or viruses (e.g., cellular or viral genomic DNA, mitochondrial DNA, chloroplast DNA, etc.).
In another aspect, the invention includes nucleic acid molecules which are purified and/or isolated by methods of the invention. In specific embodiments, the nucleic acid molecules purified and/or isolated by methods of the invention may used in molecular biological processes (e.g., may be amplified by PCR). In a related aspect, the invention is further directed to methods of making recombinant host cells comprising introducing nucleic acid molecules produced by methods of the invention.
In an additional aspect, the invention provides methods for separating RNA from DNA comprising:
(a) contacting solid matrices with samples which contain RNA and DNA;
(b) contacting solid matrices of (a) with washing solutions under conditions sufficient to remove the RNA while the DNA is retained (for example, by washing the solid matrices for periods of time ranging from 1 second to 90 minutes); and
(c) contacting the washed solid matrices with releasing reagents comprising one or more alkanol amines, under conditions which favor release of the DNA from the solid matrices.
In yet another aspect, the invention provides methods for separating closed, circular nucleic acid molecules from linear nucleic acid molecules comprising:
(a) contacting solid matrices with samples which contain closed, circular nucleic acid molecules and linear nucleic acid molecules;
(b) contacting solid matrices of (a) with washing solutions under conditions sufficient to remove the closed, circular nucleic acid molecules while the linear nucleic acid molecules are retained (for example, by washing the solid matrices for periods of time ranging from 1 second to 90 minutes) and
(c) contacting the washed solid matrices with releasing reagents comprising one or more alkanol amines, under conditions which favor release of the linear nucleic acid molecules from the solid matrices.
In another aspect, the invention further provides methods for separating nucleic acid molecules on the basis of size comprising:
(a) contacting solid matrices with samples which contain nucleic acid molecules of different sizes;
(b) contacting solid matrices of (a) with washing solutions under conditions sufficient to remove smaller nucleic acid molecules while larger nucleic acid molecules are retained (for example, by washing the solid matrices for periods of time ranging from 1 second to 90 minutes); and
(c) contacting the washed solid matrices with releasing reagents comprising one or more alkanol amines, under conditions which favor release of the larger nucleic acid molecules from the solid matrices.
In specific embodiments, washing solutions used in methods of the invention comprise 10 mM Tris-HCl, 1 mM EDTA (pH 7.3), water, or FTA(copyright) Purification Reagent (Invitrogen Corp., Life Technologies Division, Cat. No. 10876-019). In related specific embodiments, these washing solutions further comprise one or more detergents.
In further specific embodiments, solid matrices are washed for a time period selected from the group consisting of about 1 second, about 3 seconds, about 5 seconds, about 20 seconds, about 30 seconds, about 45 seconds, about 1 minute, about 5 minutes, about 10 minutes, and about 30 minutes.
In additional specific embodiments, when nucleic acid molecules are separated from each other on the basis of size, nucleic acid molecules having an average size of from about 1 kilobase to about 50 kilobases are separated from nucleic acid molecules having an average size of from about 100 kilobases to about 1,000 kilobases; nucleic acid molecules having an average size of from about 50 kilobase to about 100 kilobases are separated from nucleic acid molecules having an average size of from about 250 kilobases to about 500 kilobases; or nucleic acid molecules having an average size of from about 50 kilobase to about 100 kilobases are separated from nucleic acid molecules having an average size of from about 1,000 kilobases to about 4,000 kilobases.
The invention further provides compositions comprising nucleic acid molecules, solid matrices, and releasing reagents, wherein the releasing reagents comprises one or more alkanol amines.
The present invention also relates to kits for carrying out methods of the invention, as well as for preparing compositions of the invention. Thus, in one general aspect, the invention provides kits for removing nucleic acid molecules from solid matrices, the kits comprising (1) one or more releasing reagents of the invention and (2) one or more components selected from the group consisting of:
(a) at least one solid matrix;
(b) at least one apparatus for applying samples to solid matrices;
(c) at least one apparatus for cutting solid matrices into sections which contain samples; and
(d) at least one washing solution.
In specific embodiments, the apparatus for applying samples to solid matrices comprises a pipette (e.g., PIPETEMAN(trademark) Models P-2, P-10, P-20, P-100, P-200, etc., Rainin Instrument Company, Inc. Rainin Road, Box 4026, Woburn, Mass., 01888). In a related embodiment, the apparatus for applying samples to the solid matrix (e.g., a hammer, device which employs a piston, etc.) results in the samples being crushed into the surface of the solid matrix. An apparatus of this type will be especially useful when the sample comprises material obtained from a plant. In another related specific embodiment, the sample application apparatus is capable of applying multiple samples to solid matrices at one time.
In additional embodiments, the apparatus for cutting solid matrices into sections produces pieces of the matrices which are of varying shapes (e.g., circular, square, rectangular, irregular, etc.).
In further additional specific embodiments, washing solutions of kits of the invention comprise 10 mM Tris-HCl, 1 mM EDTA (pH 7.3), water, or FTA(copyright) Purification Reagent (Invitrogen Corp., Life Technologies Division, Cat. No. 10876-019). In related specific embodiments, washing solutions of kits of the invention further comprise one or more detergents.
Other embodiments of the invention will be apparent to one of ordinary skill in light of what is known in the art, the following drawings and description of the invention, and the claims.